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RESEARCH  CONTRIBUTIONS

 
     
 


DNA transport mechanism of type-2A topoisomerases

The "three-gate" mechanism


The capture of a DNA double helix by an ATP-dependent protein clamp: A key step in DNA transport by type II DNA topoisomerases.
  Joaquim Roca and James C. Wang.
Cell 71, 833-840. (1992).


DNA transport by a type II DNA topoisomerase: Evidence in favor of a two-gate mechanism.
 Joaquim Roca and James C. Wang.
Cell 77, 609-616. (1994).


On the Simultaneous Binding of Eukaryotic Topoisomerase II to a Pair of Double-Stranded DNA.
  Joaquim Roca, James Berger, and James Wang.
J. Biol. Chem. 268,  14250-14255. (1993).


Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp.
 Joaquim Roca, Ishida R,  Berger J. M.,  Andoh T,  and  Wang C.
Proc. Natl. Acad. Sci. USA  91, 1781-1785. (1994).


DNA transport by type II DNA topoisomerases: Direct evidence of a two gate mechanism.
 Joaquim Roca, James Berger, Sephen Harrison, and James Wang.
Proc. Natl. Acad. Sci. USA 93, 4057 - 4062,  (1996).


The path of the DNA along the dimer interface of topoisomerase II.
 Joaquim Roca.
J. Biol. Chem. 279,  25783–25788 (2004).

Topo II DNA cross

Type-2 topoisomerases "invert" DNA-crossings

G-T seg Roca©

A G-segment is trasiently gated to allow passage of a T-segment

 

T2 IC ROCA©

T-segments traverse the entire dimer interface of type-2A topoiosomerases by crossing the entry N-gate, the DNA-gate and the exit C-gate

     
 


DNA transport preferences of topoisomerase II 


DNA crossings geometry determine T-segment capture probability


The probabilities of supercoil removal and decatenation by yeast DNA topoisomerase II.
 Joaquim Roca  and  James Wang.
Genes to Cells 1,   17 - 27  (1996)


Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA.  
Joaquim Roca.
J. Mol. Biol. 305, 441-450, (2001)


Asymmetric removal of supercoils suggests how topoisomerase II simplifies DNA topology.
  Sonia Trigueros, Javier Salceda, Ignacio Bermúdez, Xavier Fernández and Joaquim Roca.
J. Mol. Biol. 335, 723-731 (2004)

 

 

TOPO II proof-reads DNA topology before completing DNA transport


Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage.
  Belen Martinez-Garcia, Xavier Fernandez, Ofelia Diaz-Ingelmo, Antonio Rodriguez-Campos, Chaysavanh Manichanh and Joaquim Roca.
Nucleic Acids Res 42: 1821-30. (2014)

cover 3 seg
DNA juxtaposition angles and loop contours in (+) and (-) supercoils

3 SEG ROCA©
Topo interaction with triple DNA crossings and DNA transport outcome
(example of a single catenane within a - and + supercoiled ring )

Proofreadin mechanims of topo II
The C-gate enables proofreading DNA topology
before T-segmetn transport is completed

     
 

Topoisomerase II  inhibitors  &  poisons

The mechanism of bisdioxopiperazines (ICRF-193)

Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp.  Joaquim Roca, Ishida R,  Berger J,  Andoh T,  and  Wang, J.
Proc. Natl. Acad. Sci. USA  91, 1781-1785. (1994)


Cloning, functional analysis and post-transcriptional regulation of a type II DNA Topoisomerase from Leishmania infantum. A new potential target for anti-parasite drugs.
Tobias Hanke, María J.Ramiro, Sonia Trigueros,  Joaquim Roca and Vicente Larraga.
Nucleic. Acids. Res.  31, 4917-4928  (2003)


Poisoning topoisomerase II   (From Roca, Thesis 1988)

     
 


Topoisomerase activity at eukarytic chromatin

 

DNA topoisomerase II activity in nonreplicating, transcriptionally inactive, chicken late spermatids.  Joaquim Roca and Cristobal Mezquita.
EMBO Journal. 8, p 1855-1860. (1989)


Circular  minichromosomes become highly recombinogenic in topoisomerase-deficient yeast Cells.
 Sonia Trigueros and Joaquim Roca.
J. Biol. Chem. 276,  2243-2249  (2001)


Failure to relax negative supercoiling of DNA  is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells. Sonia Trigueros and Joaquim Roca
J. Biol. Chem. 277, 37207-37221  (2002)


A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II.  Sonia Trigueros and Joaquim Roca.
Genes to Cells 7, 249-257 (2002)


Topoisomerase II is required for the production of long Pol II gene transcripts in yeast. Ricky Joshi, Benjamin Piña and Joaquim Roca.
Nucleic Acids Res. 40: 7907-7915 (2012)


Topoisomerase II regulates yeast genes with singular chromatin architectures. Nikolaou C, Bermúdez I, Manichanh C, García-Martinez J, Guigó R, Pérez-Ortín JE and Roca J.
Nucleic Acids Res. 41, 9243-9256 (2013)

 

 

Tunning DNA torsional stress at eukaryotic chromatin


Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. Javier Salceda, Xavier Fernández, and Joaquim Roca.
EMBO Journal 25, 2575-2583 (2006)



Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils. Xavier Fernandez, Ofelia Diaz-Ingelmo, Belen Martinez-Garcia, Joaquim Roca.
EMBO Journal  33: 1492-501(2014)

 


T2xL
TOPO II-mediated relaxation of (+) DNA supercoils allows proper elongation of long gene transcripts


T2-prom
Distintive promoter architecture of genes regulated by TOPO II


DNA Model 2006

The "cross-inversion" mechanism of TOPO II relaxes chromatin more efficiently than the "strand rotation" mechanism of TOPO I


Unbalanced relaxation of TS Chromatin conformation modulates TOPO I & TOPO II efficiency,
which results in an unbalanced relaxation of (-) and (+) torsional stress

     
 


Deciphering the 3D path of DNA by KNOT analysis

DNA packaging in viral capsids


Novel display of knotted DNA molecules by two-dimensional gel electrophoresis.
Sonia Trigueros,  Javier Arsuaga,  Maria E. Vazquez, De Witt Sumners and Joaquim Roca.
Nucleic. Acids. Res.  29, 13 - e67  (2001)


Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in Phage capsids. Javier Arsuaga, Mariel Vazquez, Sonia Trigueros, De Witt Sumners, and Joaquim Roca
Proc. Natl. Acad. Sci. USA 99, 5373-5377  (2002)


DNA knots reveal a chiral organization of DNA in phage capsids. Javier Arsuaga, Mariel Vazquez, Paul McGuirk, Sonia Trigueros, De Witt Sumners and Joaquim Roca.
Proc. Natl. Acad. Sci. USA  102, 9165-9169  (2005)


Fractal Dimension of DNA Knots. Erika Ercolini, Francesco Valle, Jozef Adamcik, Ralf Metzler, Paolo De Los Rios, Joaquim Roca and Giovanni Dietler. 
Physical Review Letters 98, 058102:1-4, (2007)


Production of highly knotted DNA by means of cosmid circularization inside phage capsids. Sonia Trigueros and Joaquim Roca.
BMC Biotechnology 7:94 (2007)

 

DNA knot 2
Display of DNA knot complexity by 2D gel electrophoresis

P4 Hyp
Distinct DNA paths produce distinct knot spectrums

DNA Knot 1
DNA packaging in viral capsids produce mainly toroidal knots

     
 

TOPOLOMICS

The "unconstrained topolome"

Genome-wide analysis of unconstrained DNA twist and writhe

 

Genome-wide analysis of chromosomal DNA topology by means of psoralen-DNA photobinding. Ignacio Bermúdez, José García-Martínez, José Pérez-Ortín, Joaquim Roca.
Nucleic Acids Res  38, e182  (2010)

 

Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes. Ricky Joshi , Benjamin Piña, and Joaquim Roca.
EMBO Journal  29, 740–748  (2010)

Psoralen 1

Psoralen-DNA phobinding maps DNA topology alterations across specific chromosomal domains

Model 2010Torsional stress rised in chromosomal DNA can dissipate by telomeres

     
 

 

The "constrained topolome"

Genome-wide analysis of constrained DNA twist and writhe

 

DNA Topology and Global Architecture of Point Centromeres. Oelia Diaz-Ingelmo, Belen Martinez-Garcia, Joana Segura, Antonio Valdes, and Joaquim Roca.
Cell Reports 13: 667-77 (2015)

Yeast point centromere

Yeast point-centromeres contraint a positive supercoil (Wr +0.6)

     
 

Methods Developed  &  Novel Approaches


Separation of rooster spermatogenic nuclei by means of centrifugal elutriation. Jacint Boix  and  Joaquim Roca.
Cytometry  14, 465-467. (1993)

A hit-and-run system for targeted genetic manipulations in yeast. Joaquim Roca, Marc Gartenberg, Yasiji Oshima and James Wang.
Nucleic Acids Res.  20, p4671-4672. (1992)

Two-dimensional agarose gel electrophoresis of DNA topoisomers. Ryo Hanai and Joaquim Roca.
Methods in Molecular Biology. Vol 94. DNA Topoisomerase Protocols. Humana Press Inc. Totowa, NJ. Ch 4, 19-27 (1999)

Filter binding assays for topoisomerase-DNA complexes. Joaquim Roca.
Methods in Molecular Biology. Vol 95. DNA Topoisomerase Protocols.  Humana Press Inc. Totowa, NJ. Ch 8, 75-80 (2001)

Two-Dimensional Agarose Gel Electrophoresis of DNA Topoisomers. Joaquim Roca.
Methods in Molecular Biology  582, pp 27-37  (2009)

 

     
 

Reviews & Comments

Joaquim Roca. The mechanism of DNA topoisomerases.
TiBS  20, 133 - 168. (1995)

Joaquim Roca. Topoisomerase II: A fitted mechanism for the chromatin landscape
Nucleic. Acids. Res. . vol  37: 721-730  (2009)

Joaquim Roca. Transcriptional inhibition by DNA torsional stress
Transcription  2, 82-85  (2011)

Joaquim Roca. The torsional state of DNA within the chromosome
Chromosoma  120: 323-34  (2011)

Joaquim Roca. In silico, In vitro and In vivo Imageries of Type II Topoisomerases
Phys Life Rev Jul 8. pii: S1571-0645(16)30060-4 (2016)

 

 
     
 

ONGOING RESEARCH PROJECTS

 

"GUIDE TOPO II"
Study the regulation of the multiple roles of topoisomerase II.

 

"DNA TANGLES"
Study of DNA knots and catenanes to infer the 3D path of intracellular DNA.

 

"TOPOLOMICS"
Genome-wide analysis of constrained and unconstrained DNA topology.

 

 

 

 

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